Flow cytometry is used to differentiate various types of cells and other similar small particles. Conventional flow cytometers commonly comprise an optically-transparent flow cell, usually made of quartz, having a central channel through which a stream of cells to be individually identified is made to flow. Movement of the cell stream through the flow cell channel is hydrodynamically entrained to the central longitudinal axis of the flow cell channel by a cell-free sheath liquid that concentrically surrounds the cell stream and flows along with the cell stream as it passes through the flow cell channel. As each cell passes through a cell-interrogation zone of the flow cell channel, it is irradiated with a focused beam of radiation (e.g., laser). Upon impinging upon each cell, the laser beam is scattered in a pattern characteristic of the morphology, density, refractive index and size of the cell. Further, the spectral characteristics of the laser beam may act to excite certain fluorochromes associated with selected cells, as may be the case when a cell's DNA has been previously stained with such fluorochromes, or when a fluorochrome molecule has been conjugated with a selected type of cell, either directly or via an intermediate. Photodetectors strategically positioned about the optical flow cell serve to convert the light-scattered by each cell and the fluorescence emitted by the excited fluorochromes to electrical signals which, when suitably processed, serve to identify the irradiated cell. In addition to the light scatter and fluorescence measurements made on each cell, some flow cytometers further characterize each cell by measuring certain physical and/or electrical properties of each cell as it passes through the flow cell. The cells may be sorted to selectively remove and collect certain cells of interest (e.g., abnormal cells) from the cells that have already passed through the optical flow cell and have been identified. Various sorting techniques have been developed, including methods requiring forming and deflecting droplets containing one or a small number of cells.
For example, a cell-sorting component may include a piezoelectric device that acts to vibrate the flow cell to produce a stream of droplets from the cell-entraining sheath liquid exiting from the flow cell. Ideally, each droplet contains but a single cell that has been characterized as to cell type by the light-scatter and fluorescence measurements just made on such cell. Each droplet in the droplet stream is then electrostatically charged as it passes between a pair of electrically charged plates, and each charged droplet is selectively deflected (or not deflected) towards a collection container as it passes between a pair of electrostatically charged deflection plates, such plates being charged to a droplet-deflecting polarity only at a time to deflect droplets (and cells) of interest. The instantaneous polarity of the deflection plates is determined by a cell-characterization processor that processes the cell-measurement signals from the optical flow cell.
Such sorting of microparticles such as cells is very important in biological research and medical applications. The alternatively to flow cytometry is microfluidic sorting. Cells are flowed through a microfluidic channel in a single file while irradiated with a focused beam of radiation (e.g., laser). Fluorescent signals of the cell are detected by photodetectors. Cells are sorted into different microfluidic channel by some kind of physical force, such as by a gas impulse (e.g., U.S. Pat. No. 4,175,662, U.S. Patent Application Publication No. 2011/0030808), by an impulsive hydraulic force created by piezoelectric beam (e.g., U.S. Pat. No. 7,392,908), or by magnetostrictive gates (e.g., U.S. Pat. No. 7,160,730), by optical force (e.g., U.S. Pat. Nos. 8,426,209, 7,745,221, 7,428,971, U.S. Patent Application Publication No. 2008/0138010), by acoustic force (e.g., U.S. Pat. No. 8,387,803, U.S. Patent Application Publication No. 2013/0192958, U.S. Patent Application Publication No. 2012/0160746), by magnetic force (e.g., U.S. Pat. Nos. 8,071,054, 7,807,454 6,120,735, 5,968,820, 5,837,200), or by dielectrophoretic force (e.g., U.S. Pat. Nos. 8,454,813, 7,425,253, 5,489,506, U.S. Patent Application Publication No. 2012/0103817). In these examples, cells never leave microfluidic channel. In contrast to conventional flow cytometer which cells are sorted in the air (air sorting), microfluidic sorting is a sorting in fluidic flow (flow sorting). All microfluidic sorting devices run at much low pressure as compared to conventional flow cytometer. They are therefore gentler to cells. Also, microfluidic sorting devices in general are less complex and less expensive than conventional flow cytometer. However, most microfluidic sorting devices sorts at a rate which is typically two or more orders of magnitude lower than that of conventional flow cytometer. Recently, a next-generation microfluidics sorting apparatus using a high-frequency fluidic valve made of a silicon microchip, which can sort as fast as conventional flow cytometer (or potentially even higher than conventional flow cytometers) has been described (U.S. Patent Application Publication No. 2015/0367346).
Because microfluidic sorting is flow sorting. It does not generate droplet. Therefore, it cannot be used to capture single cell. In addition, the vast majority of known cell sorting mechanisms are focused on the sorting mixed cells into two or more populations. Droplet sorting by electrostatic force as described in U.S. Pat. No. 3,710,933 is the preferred mechanisms to deliver sorted individual cell to a predetermined location in real time. Unfortunately, droplet sorting is typically limited to delivery of sorted individual cells to a relative large area (e.g., more than 5 mm in diameter); for areas smaller than 5 mm in diameter, the deliver accuracy becomes very low because droplets typically travel at speed more than 1 m/s and to aim the droplets precisely to an area less 5 mm in diameter (e.g., using electrostatic force) is very difficult, particularly the speed of droplet is not constant. For example, currently available droplet cell sorters, such as BD ARIA III can sort individual cells directly into 96-well cell culture plate, in which the area of each well is about 6.5 mm in diameter, with accuracy of 70%. However, sorting individual cell into 384-well cell culture plate which is about 3 mm in diameter is not practical.
In contrast, there are commercially available technologies for delivering small volumes of liquids to precise locations. For example, the FLEXDROP (Perkin Elmer) is a liquid dispenser capable of delivering small amounts of liquid to a precise location having an area of less than 1 mm in diameter. Unfortunately, such liquid dispensers cannot sort cells.
Conventional systems that use flow cells or cartridges for processing microparticles typically rely on a single reusable cartridge for processing samples. Only after a certain amount of time is a sample cartridge replaced. This often means that a single cartridge processes hundreds if not thousands of samples before being replaced. In some instances, the flow cells or cartridges are never replaced until they fail. The reusable cartridges include a multitude of microliter or less volume channels, ports for connecting to the rest of the flow cytometry system, and reservoirs for retaining the sample as well as in some cases solvents, buffers, and/or biological media. In conventional systems, components including cartridges have to be flushed out prior to loading a new sample. While a flush cycle will remove the majority of sample and fluid medium from prior runs, over time residues may still build up not only within the channels but also along connection ports, inlets, and outlets. Having residue buildup within the cartridge may lead to inaccurate application of fluid flow through the cartridge channels. Residue buildup within the channels may also result in inaccurate microparticle detection and sorting. In addition to increased risk of contamination and decreased accuracy of sorting over time, current flow cytometry demands include processing large number of samples where washing the cartridge after each run may add up to large amount of time lost.
It would be advantageous to provide microfluidic sorting methods and apparatuses that may be used to sort both groups of separate microparticles and individual microparticles, rapidly and efficiently. It would also be useful to provide disposable cartridges for sorting that would eliminate the need for cleaning the sorting cartridge after each use. It would further be advantageous if the disposable cartridge sorter possessed a simple design that was also cost effective to manufacture in large quantities which make it feasible for the cartridges to be a one-time use only component of the microparticle sorting process.